Dopaminergic neurons in rat ventral midbrain express brain-derived neurotrophic factor and neurotrophin-3 mRNAs

Studies of the trophic activities of brain-derived neurotrophic factor and neurotrophin-3 indicate that both molecules support the survival of a number of different embryonic cell types in culture. We have shown that mRNAs for brain-derived neurotrophic factor and neurotrophin-3 are localized to specific ventral mesencephalic regions containing dopaminergic cell bodies, including the substantia nigra and ventral tegmental area. In the present study, in situ hybridization with ³⁵S-labeled cRNA probes for the neurotrophin mRNAs was combined with neurotoxin lesions or with immunocytochemistry for the catecholamine-synthesizing enzyme tyrosine hydroxylase to determine whether the dopaminergic neurons, themselves, synthesize the neurotrophins in adult rat midbrain. Following unilateral destruction of the midbrain dopamine cells with 6-hydroxydopamine, a substantial, but incomplete, depletion of brain-derived neurotrophic factor and neurotrophin-3 mRNA-containing cells was observed in the ipsilateral substantia nigra pars compacta and ventral tegmental area. In other rats, combined in situ hybridization and tyrosine hydroxylase immunocytochemistry demonstrated that the vast majority of the neurotrophin mRNA-containing neurons in the substantia nigra and ventral tegmental area were tyrosine hydroxylase immunoreactive. Of the total population of tyrosine hydroxylase-positive cells, double-labeled neurons constituted 25–50% in the ventral tegmental area and 10–30% in the substantia nigra pars compacta, with the proportion being greater in medial pars compacta. In addition, tyrosine hydroxylase/neurotrophin mRNA coexistence was observed in neurons in other mesencephalic regions including the retrorubral field, interfascicular nucleus, rostral and central linear nuclei, dorsal raphe nucleus, and supramammillary region. The present results demonstrate brain-derived neurotrophic factor and neurotrophin-3 expression by adult midbrain dopamine neurons and support the suggestion that these neurotrophins influence dopamine neurons via autocrine or paracrine mechanisms. These data raise the additional possibility that inappropriate expression of the neurotrophins by dopaminergic neurons could contribute to the neuropathology of disease states such as Parkinson's disease and schizophrenia. © 1994 Wiley-Liss, Inc.     

Expression of neuronal nicotinic acetylcholine receptor subunit mRNAs within midbrain dopamine neurons

Many behavioral effects of nicotine result from activation of nigrostriatal and mesolimbic dopaminergic systems. Nicotine regulates dopamine release not only by stimulation of nicotinic acetylcholine receptors (nAChRs) on dopamine cell bodies within the substantia nigra and ventral tegmental area (SN/VTA), but also on presynaptic nAChRs located on striatal terminals. The nAChR subtype(s) present on both cell bodies and terminals is still a matter of controversy. The purpose of this study was to use double-labeling in situ hybridization to identify nAChR subunit mRNAs expressed within dopamine neurons of the SN/VTA, by using a digoxigenin-labeled riboprobe for tyrosine hydroxylase as the dopamine cell marker and (35)S-labeled riboprobes for nAChR subunits. The results reveal a heterogeneous population of nAChR subunit mRNAs within midbrain dopamine neurons. Within the SN, almost all dopamine neurons express alpha2, alpha4, alpha5, alpha6, beta2, and beta3 nAChR mRNAs, with more than half also expressing alpha3 and alpha7 mRNAs. In contrast, less than 10% express beta4 mRNA. Within the VTA, a similar pattern of nAChR subunit mRNA expression is observed except that most subunits are expressed in a slightly lower percentage of dopamine neurons than in the SN. Within the SN, alpha4, beta2, alpha7, and beta4 mRNAs are also expressed in a significant number of nondopaminergic neurons, whereas within the VTA this only occurs for beta4. The heterogeneity in the expression of nAChR subunits within the SN/VTA may indicate the formation of a variety of different nAChR subtypes on cell bodies and terminals of the nigrostriatal and mesolimbic pathways.     

Differential effects of growth/differentiation factor 5 and glial cell line-derived neurotrophic factor on dopaminergic neurons and astroglia in cultures of embryonic rat midbrain

Parkinson's disease is characterized by the progressive degeneration of midbrain dopaminergic neurons. Several studies have examined the effects of the dopaminergic neurotrophins growth/differentiation factor 5 (GDF5) and glial cell line-derived neurotrophic factor (GDNF) on these neurons in vitro. However, there is little information regarding their effects on astroglial cells. Here, the effects of GDF5 and GDNF on dopaminergic neuronal and astroglial survival and differentiation in embryonic rat midbrain cultures were examined. Both GDF5 and GDNF enhanced the survival and differentiation of dopaminergic neurons. GDF5 significantly increased the survival of astroglial cells, whereas GDNF had no significant effect on these cells. The possible involvement of astroglia in the dopaminergic neurotrophic effect induced by GDF5 was investigated by examining the effect of GDF5 on the survival of dopaminergic neurons in glia-depleted midbrain cultures. There was no significant difference between the survival of dopaminergic neurons in glia-depleted cultures treated with GDF5 and that in mixed cell cultures treated with GDF5, suggesting that GDF5 acts directly on dopaminergic neurons in exerting its neurotrophic effect. GDF5 and GDNF have been established as potent neurotrophic factors for dopaminergic neurons. However, the effects of adding a combination of these neurotrophins to midbrain cultures have not been previously examined. The present study found that combined treatment with GDF5 and GDNF significantly increased the survival of dopaminergic neurons in cultures compared with that in cultures treated with either neurotrophin alone. This was an additive effect, indicating that these neurotrophins act on separate subpopulations of dopaminergic neurons.     

Inverse relationship between the contents of neuromelanin pigment and the vesicular monoamine transporter-2: human midbrain dopamine neurons

The dopaminergic neurons in the ventral substantia nigra (SN) are significantly more vulnerable to degeneration in Parkinson's disease (PD) than the dopaminergic neurons in the ventral tegmental area (VTA). The ventral SN neurons also contain significantly more neuromelanin pigment than the dopaminergic neurons in the VTA. In vitro data indicate that neuromelanin pigment is formed from the excess cytosolic catecholamine that is not accumulated into synaptic vesicles by the vesicular monoamine transporter-2 (VMAT2). By using quantitative immunohistochemical methods in human postmortem brain, we sought to examine the relative contents of VMAT2 within neurons that contain different amounts of neuromelanin pigment. The immunostaining intensity (ISI) was measured for VMAT2 and also for the rate-limiting enzyme for the synthesis of dopamine, tyrosine hydroxylase (TH). ISI measures were taken from the ventral SN region where neurons are most vulnerable to degeneration in PD, nigrosome-1 (N1); from the ventral SN region where cells are moderately vulnerable to degeneration in PD, the matrix (M); and from VTA neurons near the exit of the third nerve (subregion III). The data indicate that 1) subregion III neurons have significantly higher levels of VMAT2 ISI compared with N1 neurons (more than twofold) and M neurons (45%); 2) there is an inverse relationship between VMAT2 ISI and neuromelanin pigment in the N1 and III neurons; 3) there is an inverse relationship between VMAT2 ISI and the vulnerability to degeneration in PD in the N1, M, and III subregions; and 4) neurons with high VMAT2 ISI also have high TH ISI. These data support the hypothesis that midbrain dopaminergic neurons that synthesize greater amounts of dopamine have more vesicular storage capacity for action potential-induced release of transmitter and that the ventral SN neurons accumulate the most neuromelanin pigment, in part because they have the least VMAT2 protein.     

Tyrosine hydroxylase-immunoreactive neurons are decreased in number in the cerebral cortex of Parkinson's disease

Tyrosine hydroxylase (TH)-immunoreactive (IR) neurons and their relationship with Lewy bodies were investigated in Parkinson's disease. Using anti-TH and/or anti-ubiquitin antibodies, we evaluated the cerebral cortices included superior frontal gyrus, precentral gyrus, postcentral gyrus, inferior temporal gyrus, cuneus, cingulate gyrus, short and long gyri of insula, and parahippocampal gyrus from 18 autopsy cases of Parkinson's disease and 16 controls. The appearance of TH-IR neurons in cerebral cortices was suggestive of non-pyramidal interneurons. The mean number of TH-IR neurons and the density of TH-IR fibers in Parkinson's disease were decreased in comparison with the controls. The cognitive impairment in Parkinson's disease has been accounted for by the lesions of the basal nucleus of Meynert, the locus ceruleus, and the cortical Lewy bodies in the cerebral cortex. In addition to these lesions, the global loss of non-pyramidal TH-IR cortical neurons and TH-IR fibers would induce the dysfunction of higher-order control of the neocortex and the limbic system in Parkinson's disease. Double-immunostaining with anti-TH and anti-ubiquitin antibodies did not show the TH-IR neuron with cortical Lewy body in the cerebral cortices of Parkinson's disease. In the cerebral cortices of Parkinson's disease, TH-IR non-pyramidal neurons in which cortical Lewy body is not formed decreased in number.     

Relationship between NMDA receptor expression and MPP+ toxicity in cultured dopaminergic cells

It has been suggested that excitotoxicity could be contributing to dopamine cell loss after methylphenylpyridinium ion (MPP+) exposure, although the literature regarding this is contradictory. Given that in cell culture excitotoxicity has been reported to be dependent on culture age, we postulated that these discrepant results might be explained by a difference in developmental expression of N-methyl-D-aspartate (NMDA) receptors. To test this, mesencephalic cells were cultured and the number of dopaminergic neurons (tyrosine hydroxylase-immunoreactive cells [TH-IR] cells) expressing the NMDA R1 subunit (NR1) was determined using double-label immunofluorescence microscopy. An increase in the percentage of TH-IR cells expressing NR1 occurred over time in culture and this correlated with the toxicity of NMDA. At 7 days in vitro (DIV 7), only 17% (n=167 cells/4 experiments) of TH-IR cells expressed NR1 and these cells were insensitive to NMDA toxicity. This increased to 80% (n=254 cells/6 experiments) by DIV 11 and cultures were now susceptible to NMDA-induced injury. Cultures grown for either 7 or 11 days were treated for 48 hr with increasing concentrations of MPP= (0.5-20 microM) and the loss of dopaminergic neurons was determined by cell counting. Cultures at DIV 7 were more sensitive to MPP= than 11-day-old cultures (LD50= approximately 0.75 microM vs. 15 microM, respectively). Co-exposure to MK-801 (5 microM) did not protect against MPP+ toxicity in young cultures, but attenuated MPP+ toxicity in the older cultures, becoming statistically significant at 20 microM MPP+. These data indicate that the activation of NMDA receptors is not required for, but can contribute to, MPP(+)-induced neurodegeneration of dopaminergic cells in culture.     

Inhibition of dopamine and choline acetyltransferase concentrations in rat CNS neurons by rat α1- and α2-macroglobulins

Previous studies have implicated human alpha-2-macroglobulin (α₂M) as a potential regulator of neuronal development and function. Rat alpha-1-macroglobulin (α₁M) and acute-phase alpha-2-macroglobulin (α₂M) are murine homologues of human α₂M. In this report, we tested the effect of intracranially infused serotonin-activated rat α₁M (5HT-α₁M) on the concentration of dopamine (DA) in the corpus striatum in vivo and the effect of 5HT-activated rat α₁M and α₂M on the choline acetyltransferase (ChAT) activity upon embryonic basal forebrain neurons in culture. The results show that direct infusion of 0.65 nmole rat 5HT-α₁M into the adult rat corpus striatum produced a consistent attenuation upon striatal DA concentrations. This decrease was particularly prominent at 5–7 days post-infusion. In addition, rat 5HT-α₁M and rat 5HT-α₂M, like human 5HT-α₂M, all significantly inhibited ChAT activity of embryonic rat cerebral cortex neurons. Although normal human α₂M and rat α₂M were either marginally or insignificantly inhibitory in this preparation, normal rat α₁M dose-dependently inhibited ChAT activity. These results demonstrate that monoamine-activated α-macroglobulins from rat depress dopaminergic and cholinergic neurotransmitter systems in the CNS, and this suggests a potential regulatory role of these alpha-macroglobulins in neurotransmitter metabolism. J. Neurosci. Res. 51:541–550, 1998. © 1998 Wiley-Liss, Inc.     

Differential modification of dopamine transporter and tyrosine hydroxylase mRNAs in midbrain of subjects with parkinson's,alzheimer's with parkinsonism,and alzheimer's disease

The molecular characteristics of midbrain dopamine (DA) neurons have been extensively studied in Parkinson's disease (PD). No such studies of the characteristics of midbrain DA neurons in Alzheimer's disease (AD) or Alzheimer's disease with parkinsonism (AD/Park) have been published. We examined the levels of tyrosine hydroxylase (TH) protein, and the expression of TH and dopamine transporter (DAT) mRNAs, in midbrain neurons of PD, AD, and AD/Park cases. In PD, the loss of TH protein in the ventral tier of the substantia nigra pars compacta (SNpc) of the PD group is accompanied by severe losses in the number of neurons that express TH mRNA and DAT mRNA (74% loss). Remaining neurons show a shift to higher concentrations of TH mRNA but a shift to lower concentrations of DAT mRNA per cell. Hence, there is evidence that compensation in the remaining neurons can elevate concentrations of TH mRNA and lower DAT mRNA. Alternatively, there may be a predilection for a loss of neurons with high levels of DAT mRNA and low TH mRNA levels within the SNpc of PD cases. There was no change in TH protein but an elevation of TH mRNA concentrations per neuron without any change in concentrations of DAT mRNA in the AD group. The AD/Park group did not exhibit changes in the level of TH protein, but showed a small loss (26%) of neurons in the SNpc and a greater loss in other regions of the midbrain (43–53%). Remaining DA neurons showed a marked shift to lower concentrations of DAT mRNA per neuron and a nonsignificant shift in cellular concentration of TH mRNA to higher levels. This is consistent with our previous work showing that with AD/Park there is a significant reduction in the number of DAT sites located on DA terminals in the striatum, but the midbrain neurons have not died. Our results indicate that the differential regulation of mRNAs encoding TH and DAT is similar in the parkinsonian disorders (PD and AD/Park) even though the degree of cell death is very different. This might suggest that compensatory events occur in these DA neurons in AD/Park that are similar to those in PD and that result in differential effects on mRNAs encoding TH and DAT proteins.     

Glial cell line-derived neurotrophic factor supports survival of injured midbrain dopaminergic neurons

Glial cell-lined derived neurotrophic factor (GDNF) has been shown to promote survival of developing mesencephalic dopaminergic neurons in vitro. In order to determine if there is a positive effect of GDNF on injured adult midbrain dopaminergic neurons in situ, we have carried out experiments in which a single dose of GDNF was injected into the substantia nigra following a unilateral lesion of the nigrostriatal system. Rats were unilaterally lesioned by a single stereotaxic injection of 6-hydroxydopamine (6-OHDA; 9 μg/4 μl normal saline with 0.02% ascorbate) into the medial forebrain bundle and tested weekly for apomorphine-induced (0.05 mg/kg s. c. ) contralateral rotation behavior, Rats that manifested >300 turns/hour received a nigral injection of 100 μg GDNF, or cytochrome C as a control, 4 weeks following the 6-OHDA lesion, Rotation behavior was quantified weekly for 5 weeks after GDNF. Rats were subsequently anesthetized, transcardially perfused, and processed for tyrosine hydroxylase immunohistochemistry. It was found that 100 μg GDNF decreased apomorphine-induced rotational behavior by more than 85%. Immunohistochemical studies revealed that tyrosine hydroxylase immunoreactivity was equally reduced in the striatum ipsilateral to the lesion in both cytochrome C and GDNF-injected animals. In contrast, large increments in tyrosine hydroxylase immunoreactivity were observed in the substantia nigra of animals treated with 100 μg of GDNF, with a significant increase in numbers of tyrosine hydroxylase-immunoreactive cell bodies and neurites as well as a small increase in the cell body area of these neurons. The results suggest that GDNF can maintain the dopaminergic neuronal phenotype in a number of nigral neurons following a unilateral nigrostriatal lesion in the rat.     

Intracellularly recorded response of rat striatal neurons in vitro to fenoldopam and SKF 38393 following lesions of midbrain dopamine cells

The effect of long-term (6–19 weeks) 6-hydroxydopamine-induced (6-OHDA) lesions of midbrain dopamine cells on dopamine D₁-like agonist-induced changes in the excitability of rat striatal neurons was investigated in vitro using tissue slices and intracellular recording techniques. Fenoldopam and (±)-SKF 38393 predominantly decreased excitability in control preparations including striatal neurons located contralateral to 6-OHDA injection sites and neurons obtained from rats receiving sham injections or no treatment. Fenoldopam also inhibited neurons ipsilateral to lesions of midbrain dopamine cells. (±)-SKF 33393, unlike fenoldopam, produced predominantly increases in the excitability of ipsilateral striatal neurons. Superfusion of the D₁ receptor antagonist, SCH 23390, blocked fenoldopam-induced decreases in excitability but not the (±)-SKF 38393-induced excitation of neurons ipsilateral to the lesion. Sequential application of fenoldopam and quinpirole, a D₂/D₃ receptor agonist, produced responses to both drugs in a majority of neurons. The results demonstrate that inhibitory responses to fenoldopam are mediated by D₁ receptors, while excitatory effects of (±)-SKF 38393 in the striatum ipsilateral to the lesion are apparently not dependent on D₁ receptor activation. These findings also suggest that dopamine D₁ and D2/D3 receptors are able to concurrently influence the excitability of striatal neurons in the dopamine deafferentated striatum. Similar regulation of striatal neurons in vivo may contribute to dopaminergic regulation of basal ganglia output and the ability of dopaminomimetic agents to ameliorate symptoms of dopaminergic deficiency in Parkinson's disease. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Parkinsonism without dopamine neuron degeneration in aged l‐dopa‐responsive dystonia knockin mice

Background : Recent neuroimaging studies implicate nigrostriatal degeneration as a critical factor in producing late‐onset parkinsonism in patients with l ‐dopa‐responsive dystonia‐causing mutations. However, postmortem anatomical studies do not reveal neurodegeneration in l ‐dopa‐responsive dystonia patients. These contrasting findings make it unclear how parkinsonism develops in l ‐dopa‐responsive dystonia mutation carriers. Methods : We prospectively assessed motor dysfunction, responses to dopaminergic challenge, and dopamine neuron degeneration with aging in a validated knockin mouse model bearing a l ‐dopa‐responsive dystonia‐causing mutation found in humans. Results : As l ‐dopa‐responsive dystonia mice aged, dystonic movements waned while locomotor activity decreased and initiation of movements slowed. Despite the age‐related reduction in movement, there was no evidence for degeneration of midbrain dopamine neurons. Presynaptically mediated dopaminergic responses did not change with age in l ‐dopa‐responsive dystonia mice, but responses to D1 dopamine receptor agonists decreased with age. Conclusions : We have demonstrated for the first time the co‐occurrence of dystonia and Parkinson's‐like features (mainly consisting of hypokinesia) in a genetic mouse model. In this model we show that these features evolve without dopaminergic neurodegeneration, suggesting that postsynaptic plasticity, rather than presynaptic degeneration, may contribute to the development of parkinsonism in patients with l ‐dopa‐responsive dystonia. © 2017 International Parkinson and Movement Disorder Society     

Variability in neuronal expression of dopamine receptors and transporters in the substantia nigra

Parkinson's disease (PD) patients have increased susceptibility to impulse control disorders. Recent studies have suggested that alterations in dopamine receptors in the midbrain underlie impulsive behaviors and that more impulsive individuals, including patients with PD, exhibit increased occupancy of their midbrain dopamine receptors. The cellular location of dopamine receptor subtypes and transporters within the human midbrain may therefore have important implications for the development of impulse control disorders in PD. The localization of the dopamine receptors (D1–D5) and dopamine transporter proteins in the upper brain stems of elderly adult humans (n = 8) was assessed using single immunoperoxidase and double immunofluorescence (with tyrosine hydroxylase to identify dopamine neurons). The relative amount of protein expressed in dopamine neurons from different regions was assessed by comparing their relative immunofluorescent intensities. The midbrain dopamine regions associated with impulsivity (medial nigra and ventral tegmental area [VTA]) expressed less dopamine transporter on their neurons than other midbrain dopamine regions. Medial nigral dopamine neurons expressed significantly greater amounts of D1 and D2 receptors and vesicular monoamine transporter than VTA dopamine neurons. The heterogeneous pattern of dopamine receptors and transporters in the human midbrain suggests that the effects of dopamine and dopamine agonists are likely to be nonuniform. The expression of excitatory D1 receptors on nigral dopamine neurons in midbrain regions associated with impulsivity, and their variable loss as seen in PD, may be of particular interest for impulse control. © 2013 International Parkinson and Movement Disorder Society     

Age-related accumulation of Marinesco bodies and lipofuscin in rhesus monkey midbrain dopamine neurons: relevance to selective neuronal vulnerability

Parkinson's disease (PD) is characterized by degeneration of nigrostriatal dopamine (DA) neurons. Although aging is a primary risk factor for PD, its role in DA neuron degeneration remains unknown. Neurodegeneration in PD is not uniform throughout the ventral midbrain: the ventral tier of the substantia nigra (vtSN) is most vulnerable, whereas the dorsal tier (dtSN) and ventral tegmental area (VTA) are relatively resistant. We studied young (9-10 years old), middle-aged (14-17 years old), and old-aged (22-29 years old) rhesus monkeys to identify factors potentially underlying selective vulnerability and their association with aging. We focused on markers relevant to the ubiquitin-proteasome (UPS) and lysosome systems. Unbiased stereological counting was performed on tyrosine hydroxylase-positive (TH+) neurons and TH+ neurons containing Marinesco bodies (TH+MB) or lipofuscin (TH+lipo), markers of UPS or lysosomal activity, respectively. TH+ neuron numbers were inversely correlated with advancing age specifically in the vtSN, not the dtSN or VTA. TH intensity decreased throughout the ventral midbrain with increasing age, an effect exacerbated in the vtSN. TH+MB neurons were localized in the vulnerable vtSN of old monkeys. The number of MBs per cell increased with age, and TH intensity of TH+MB neurons decreased in middle age. Conversely, TH+lipo neurons were primarily found in the resistant dtSN and VTA. These data suggest that particular age-related changes localize to DAergic subregions relevant to degenerative patterns in PD. Furthermore, the results begin to characterize the nature of the link between aging and PD, and they support the concept that aged monkeys represent a valuable model for studying specific events preceding PD.     

Methamphetamine-induced loss of striatal dopamine innervation in BDNF heterozygote mice does not further reduce D3 receptor concentrations

Depletion of dopamine (DA) reduces D(3) receptor number, but D(3) receptor expression is also regulated by brain-derived neurotrophic factor (BDNF). We took advantage of transgenic heterozygous BDNF mutant mice (+/-) to determine if reduced BDNF and loss of DA fibers produced by methamphetamine were additive in their impact on D(3) receptor number. We assessed selective markers of the dopaminergic system including caudate-putamen DA concentrations and quantitative autoradiographic measurement of tyrosine hydroxylase (TH) levels, DA transporter (DAT), and DA D(3) receptor binding between vehicle and methamphetamine-treated BDNF +/- and their wildtype (WT) littermate control mice. Caudate-putamen DA concentrations, TH and DAT levels were significantly reduced following methamphetamine treatment in both WT and BDNF +/- mice. The extent of methamphetamine-induced reduction in TH and DAT was greater for the WT than BDNF +/- mice and DAT levels were also decreased to a greater extent in nucleus accumbens of WT as compared to BDNF +/- mice. Lower D(3) receptor existed in caudate-putamen and nucleus accumbens in BDNF +/- mice and these differences were not affected by methamphetamine treatment. Taken together, these results not only substantiate the importance of BDNF in controlling D(3) receptor expression, but also indicate that a methamphetamine-induced depletion of DA fibers fails to produce an additive effect with lowered BDNF for control of D(3) receptor expression. In addition, the reduction of D(3) receptor expression is associated with a decreased neurotoxic response to methamphetamine in BDNF +/- mice.     

Generation of dopamine neurons from embryonic stem cells in the presence of the neuralizing activity of bone marrow stromal cells derived from adult mice

Stromal cell lines such as PA6 and MS5 have been employed for generating dopamine (DA) neurons from embryonic stem (ES) cells. The present study was designed to test whether bone marrow stromal cells (BMSC) derived from adult mice might be available as a feeder layer to produce DA cells efficiently from ES cells. When ES cells were grown on BMSC in the presence of fibroblast growth factor 8 (FGF8) and sonic hedgehog (SHH), about 40% of TuJ1-positive neurons expressed tyrosine hydroxylase (TH). Because these cells labeled with TH were negative for dopamine-beta-hydroxylasae (DBH), the marker for noradrenergic and adrenergic neurons, the TH-positive cells were most likely DA neurons. They indeed expressed midbrain DA neuron markers such as Nurr 1, Ptx-3, and c-ret and were capable of synthesizing and releasing DA in vitro. Furthermore, DA neurons differentiated from ES cells in this differentiation protocol survived transplantation in rats with 6-hydroxydopamine lesions and reversed the lesion-induced circling behavior. The data indicate that BMSC can facilitate an efficient induction of DA neurons from ES cells and that the generated DA neurons are biologically functional both in vitro and in vivo. Insofar as BMSC have recently been employed in autologous cell therapy for ischemic heart and arteriosclerotic limb diseases, the present study raises the possibility that autologous BMSC can be applied in future cell transplantation therapy in Parkinson's disease.     

Alterations of dopamine release by rat caudate putamen tissues superfused with α2-Macroglobulin

Monoamine-activated alpha-2-macroglobulin (α₂M) has been shown to decrease the dopamine concentrations in rat caudate putamen (CP) in vivo as well as inhibit choline acetyltransferase activities in the culture of basal forebrain neurons. In this study, we further investigated the effects of methylamine-activated α₂M (MA-α₂M) upon striatal rlopaminergic function by determining whether a direct infusion of this glycoprotein will alter dopamine (DA) release in vitro from superfused CP tissue fragments. In experiment 1, an infusion of 2.8 μM MA-α₂M produced a statistically significant increase in DA release compared with control superfusions. In experiment 2, varying doses (0, 0.7, 1.4, 2.8, 4.1 μM) of MA-α₂M were tested for their capacity to alter DA release. Only the 2.8 μM dose of MA-α₂M was effective in producing a significant increase of DA release. In experiment 3, the normal form of α₂M (N-α₂M) at 2.8 μM was compared with the control superfusions. The infusion of N-α₂M produced an increase in DA release which was substantially lower than the DA increase induced by MA-α₂M, and not significantly different from that of the control superfusion. These results show that MA-α₂M, like some other neurotoxins, can markedly alter CP dapaminergic function as indicated by the acute increase in DA release following infusion of this glycoprotein, and these effects are exerted at a relatively narrow range of doses. Taken together, these data suggest that this glycoprotein, if allowed to accumulate in the central nervous system (CNS), may promote some neurodegenerative changes that can occur in disorders like Parkinson's disease. © 1996 Wiley-Liss, Inc.